Large Scale Transgenic and Cluster Deletion Analysis of the Hoxd Complex Separate an Ancestral Regulatory Module from Evolutionary Innovations

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RESEARCH COMMUNICATION

where groups of genes acquired shared enhancer se- Large scale transgenic and quences acting independently of colinearity. For in- cluster deletion analysis of the stance, the early phase of Hoxd gene expression in limb buds is regulated in a colinear fashion, whereas expres- HoxD complexseparate an sion of the same genes in digits is concurrent, rather than ancestral regulatory module colinear (Nelson et al. 1996). In the HoxD complex, gene recruitment involved in from evolutionary innovations many instances the design of potent enhancer sequences, which regulate several genes at once. We proposed ear- François Spitz,1 Federico Gonzalez,1 lier that expression of four genes in developing digits was Catherine Peichel,2,3 Thomas F. Vogt,2,4 controlled by a unique enhancer that displays poor pro- Denis Duboule,1,5 and József Zákány1 moter specificity as it influenced foreign promoters when targeted to the locus (van der Hoeven et al. 1996; 1Department of Zoology and Animal Biology, University Hérault et al. 1999). Targeted deletions in the posterior of Geneva, Sciences III, 1211 Geneva 4, Switzerland; HoxD complex placed this enhancer somewhere up- 2Department of Molecular Biology, Princeton University, stream of Evx2, outside the cluster (Kondo and Duboule Princeton, New Jersey 08544, USA 1999). Likewise, several genes respond to a gut enhancer sequence that is required to form the ileo-coecal sphinc- The ancestral role of the Hox gene family is specifying ter (Zakany and Duboule 1999) and is localized either in morphogenetic differences along the main body axis. In the first 30 kb of the complex (around Hoxd1) or outside vertebrates, HoxD genes were also co-opted along with of the complex (Kmita et al. 2000b). the emergence of novel structures such as limbs and Regulation inside Hox clusters is complex because of a genitalia. We propose that these functional recruitments high density of genes with embedded and shared regulatory elements, making it difficult to assign a control se- relied on the appearance, or implementation, of regula- quence to one individual gene, rather than to a series of tory sequences outside of the complex. Whereas transgenes (Gérard et al. 1996;Hérault et al. 1998;Sharpe et genic human and murine HOXD clusters could function al. 1998). To assess which features of Hoxd gene regula- during axial patterning, in mice they were not expressed tion are intrinsic to the complex and which are located at outside the trunk. Accordingly, deletion of the entire a distance (i.e., act in a global scale over the locus) we cluster abolished axial expression, whereas recently ac- produced transgenic mice carrying additional HoxD loci. quired regulatory controls were preserved. We used a human PAC extending from HOXD3 to 30 kb upstream EVX2 and a mouse BAC containing a tagged Received April 12, 2001;revised version accepted July 2, 2001. HoxD. We compared the regulatory potentials of these transgenic clusters with that of targeted deletions of the During vertebrate development, Hox genes are activated mouse HoxD complex, including a knock-in replace- in a spatio-temporal sequence that leads to partially ment of the cluster. We show that both approaches overlapping transcript domains along the trunk axis. mapped regulatory elements responsible for colinear ex- These expression domains generate various combina- pression within the cluster or close to it, whereas non- tions of HOX proteins at different anterior–posterior po- colinear expression in appendicular structures is dictated sitions, instructing cohorts of cells about their fate (e.g. by regulatory elements located at remote positions. We Krumlauf 1994). In most cases, there exists a correspon- also show that the presence of the complex is required dence between the order of the genes in the genome and for the maintenance of Hox gene expression profiles their domains of expression, a phenomenon referred to as throughout development. colinearity. This feature is very ancient, as it seems to operate in all animals with a bilateral body plan;hence, Results it is likely that colinearity relies on a conserved mecha- Rescue of Hoxd mutations by a human HOXD complex nism. In addition to this function, Hox genes were recruited To study the regulatory potential of a Hox complex, we during evolution to carry out a number of other tasks. produced transgenic mice carrying a 120 kb large human For example, a given subset of vertebrate Hox genes is PAC (Fig. 1A) extending from the HOXD3–HOXD1 re- required for limb development (Davis and Capecchi gion to ∼30 kb 5Ј upstream of EVX2. Five founder ani- 1996;Rijli and Chambon 1997;Zakany et al. 1997), gut mals were recovered. One carried a partial PAC and thus morphogenesis (Zakany and Duboule 1999), or hair fol- was not studied further. Among the others, three lines licle development (Godwin et al. 1998). In some cases, with either one or two copies of the transgene were es- the colinear process was also recruited, whereas else- tablished (TgN[HOXD]1–3). One high-copy number animal was recovered but the line could not be established due to perinatal lethality. [Key Words: Hox cluster;colinearity;BAC;remote enhancers] Present addresses: 3Department of Developmental Biology, Stanford Uni- Mice with the human PAC showed abnormal vertebral versity, Stanford, CA 94305, USA; 4Department of Pharmacology, Merck formulae with a high penetrance of five lumbar vertebrae Research Laboratories, Merck & Co., West Point, PA 19486, USA. (L5) instead of the normal L6 (Fig. 2, top). Besides this 5 Corresponding author. anteriorization, no alteration was observed, suggesting E-MAIL [email protected]; FAX 41-22-702-6795. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ that the human genes were expressed during mouse gad.205701. trunk development with the appropriate specificity. This

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mouse counterparts. Analysis of human transgene expression revealed that the HOXD genes were regulated correctly during trunk development, as expression boundaries were similar to those of the corresponding resident mouse genes (Fig. 3A, top, arrows). Human HOXD13 was weakly expressed posteriorly, starting caudal to the hindlimb bud, whereas expression of HOXD11 was at the level of pv25, around the lumbo– sacral transition. As expected, HOXD4 was expressed up to the hindbrain and in somites, similar to the mouse gene. These results indicated that the rescue observed in the vertebrae derived from a faithful expression of the transgenes. In contrast, expression of human genes in developing limbs was deficient. Early on, weak expression of both HOXD13 and HOXD11 was detected in the posterior limb bud (Fig. 3A, top, arrows). At this stage, transcript distribution differed from that of the corresponding mouse genes, which gave stronger signals over a wider domain including distal parts, in addition to the posterior half (Fig. 3A, top). Subsequently (Fig. 3A, bottom), the difference accentuated with an almost complete dis- appearance of all transcripts from the limbs for both HOXD13 and HOXD11, whereas endogenous genes developed their robust expression patterns in both presumptive digits (Hoxd13 and Hoxd11) and forearms (Hoxd11). Expression of human posterior genes was also absent from the developing genitalia, whereas a strong Figure 1. The mouse HoxD cluster, the human PAC, and signal was detected for mouse Hoxd10, Hoxd11, nested deletions. (A)TheHoxD complex (top) with genes as Hoxd12, and Hoxd13. Likewise, expression of the mouse black boxes (red for human). Below is the human PAC 78j1, Hoxd4 and Hoxd11 genes in part of the intestinal hernia with the human HOXD3 to EVX2 genes and 30 kb of DNA (Fig. 3A, bottom, arrows) was not recapitulated by the upstream, and the mouse BAC 400h17, with the murine cluster PAC transgene. and 100 kb of DNA upstream. (B) Scheme of nested deletions. These observations implied that enhancers regulating (Top)TheHoxD complex with a Hoxd11/lacZ reporter trans- Hoxd genes in limbs, genitalia, and intestinal hernia gene inserted upstream of Hoxd13. After recombination of a were absent from the 120-kb human PAC. This was fur- loxP site within Hoxd1 (arrowhead), Cre-mediated deletion re- ther investigated with transgenic mice carrying a 215-kb moved the cluster leaving the reporter gene construct TgHd11/ lacDel9 (below). The bottom lines are intermediate configurations obtained by using other loxP sites within the cluster. After recombination, partial deletions of either three (Del3) or seven (Del7) genes are obtained, with the reporter transgene at the same position. was verified further when the transgene was combined with a triple inactivation in cis of Hoxd13, Hoxd12, and Hoxd11 (Zakany and Duboule 1996). Mice (16 out of 18) lacking these functions (HoxDDel3/Del3) displayed an L7 formula (a supernumerary lumbar vertebra;Fig. 2, top). The introduction of the human PAC into this back- ground reinstated the predominant L6 formula (Fig. 2, top) with a substantial incidence of L5 animals. This showed a robust rescue of the mouse defect by the human proteins. Nevertheless, rescue was not detected in the limb skeleton and no difference was scored between wild-type and transgenic hand skeletons (Fig. 2, bottom). Because human HOXD proteins could functionally rescue vertebral alterations, lack of rescue in limbs was likely due to Figure 2. Vertebral and limb phenotypes. (Top) Skeletal prepa- abnormal expression of the transgenes in these struc- rations of lumbo–sacral transitions. Wild-type mice have six tures. Thus, we analyzed the expression of human lumbar vertebrae (L6). Mice transgenic for human HOXD PAC HOXD genes during mouse development. (TgN[HOXD]) had an anteriorized lumbo–sacral transition with Human HOXD genes in the mouse L5. In contrast, mice carrying a deletion of Hoxd13 to Hoxd11 showed seven lumbar vertebra (L7). When the human HOXD We used RNA probes specific for human HOXD13, transgene was added to this latter configuration (TgN[HOXD]; HOXD11, and HOXD4 transcripts, in parallel with the Del3/Del3), the L7 phenotype was rescued to L6.

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the trunk. However, as for the human PAC, Hoxd11/ lacZ failed to be expressed in distal limbs, genitalia, and intestinal hernia. The transgenic pattern resembled that described for the short conventional transgene (Gérard et al. 1993), suggesting that no major Hoxd11 regulatory elements were localized in the 215-kb piece of DNA, other than those already present at the Hoxd11 locus.

Deletion of the mouse HoxD complex To confirm the remote locations of these enhancers, we engineered a full deletion of HoxD, from Hoxd1 to upstream Hoxd13 (Del9). This deletion was made by plac- ing a Hoxd1/lacZ fusion along with a loxP site into a chromosome carrying a Hoxd11/lacZ reporter transgene (TgH[d11/lac]Ge) between Hoxd13 and Evx2 (Fig. 1B). As this latter transgene also contains a loxP site, treatment of positive embryonic stem (ES) cells with Cre recombinase led to the replacement of the entire complex by a Hoxd11/lac reporter transgene. Once the cluster had been removed, ␤-gal detection was used as an indicator of the remaining regulatory influences. This expression (Fig. 4;Del9) was compared with that of the same transgene, either when recombined upstream of Hoxd13 (Fig. 4; d11/lac), or after partial deletions (Fig. 4;Del3 and Del7, respectively). In this way, the transcription of the same reporter gene was monitored at four different positions in the cluster, along with a progressive reduction of the gene complex. In embryonic day (E)9.5 embryos, the TgH[d11/lac]Ge transgene was severely suppressed, giving a delayed signal restricted to the posterior part of the embryo (Fig. 4A), thus resembling that of the neighboring gene Hoxd13 (van der Hoeven et al. 1996). When the trans- Figure 3. (A) Human HOXD genes in mice. In E10.5 embryos gene was placed near Hoxd10, along with a deletion of (top), the human HOXD13, HOXD11, and HOXD4 genes were Hoxd13 and Hoxd12, expression extended anteriorly, to expressed in the trunk, up to anterior levels similar to those of recapitulate the Hoxd11 pattern (Del3;Fig. 4B). After the corresponding murine genes (black arrows). Human deletion of seven genes (Del7) or of the entire complex HOXD13 and HOXD11 were also expressed weakly in the de- (Del9), the early expression profile was not much differ- veloping posterior hindlimbs (arrowheads). In contrast, mouse ent from the Del3 pattern. However, lacZ expression Hoxd13 and Hoxd11 were expressed in both hindlimb and fore- was detected at a more anterior body level, including the limb buds, with a more anterior extension (black arrowhead). In intermediate plate mesoderm and the emerging forelimb E11.5 embryos (bottom), the human genes were expressed like buds (Fig. 4C–D). Subsequently, well-established Hox their murine counterparts in the trunk. However, neither gene profiles were observed (Fig. 4E–G), with a posterior HOXD13 nor HOXD11 were expressed in the limbs or genital restriction for the TgH[d11/lac]Ge locus in both spinal bud (black arrows), whereas the murine genes were strongly cord and somitic mesoderm. A rostral extension of these active there. Arrowheads indicate the intestinal hernia, which expression domains was observed in Del3 animals, stained with both Hoxd4 and Hoxd11 probes, while the human reaching the lumbar region (Fig. 4F, black arrowhead), genes were silent. (B) Hoxd11/lacZ expression in transgenic em- whereas expression in Del7 was wider, resembling that bryo with a 215 kb mouse BAC with the entire HoxD cluster of Hoxd3 or Hoxd4. In this latter case, a “pan–Hoxd” and lacZ sequences within Hoxd11. The faint expression in pattern was recovered, in which all expression sites for posterior hindlimb did not correspond to the signal given by the Hoxd genes were stained. In developing limbs, a strong endogenous gene (cf. with Hoxd11 in A). staining was seen in the distal parts, the presumptive digits, with additional staining in the future forearm for large BAC with the murine HoxD complex, starting 100 both Del3 and Del7 configurations (Fig. 4F–G). At this kb 5Ј of Hoxd13 and extending up to 10 kb downstream stage, expression was also observed in the genital bud for of Hoxd1. Thus, it contained an additional 95 kb of all three lines, whereas only the Del3 and Del7 fetuses flanking DNA when compared to the human PAC. By displayed staining in their intestinal hernia (Fig. 4F–G). using recE/T dependent recombination (Zhang et al. The staining of Del9 embryos revealed two remarkable 1998;Muyrers et al. 1999), lacZ reporter sequences were features. First, staining was absent from most of the de- introduced in the Hoxd11 gene to follow its expression veloping CNS and somitic mesoderm of E11.5 fetuses in mice transgenic for the BAC. Three founders were (Fig. 4H). Therefore, the deletion of the cluster not only recovered, stained for ␤-gal activity and compared with removed elements necessary for colinear expression, but the conventional Hoxd11/lacZ transgene patterns also prevented expression of the Hoxd11/lacZ transgene (Gérard et al. 1993;Fig. 3B). In all cases, the BAC Hoxd11 in the posterior regions, as expected from its behavior at was expressed with the expected anterior boundary in random genomic positions (Gérard et al. 1993). Second,

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However, a diffuse staining appeared throughout Del7 embryos, including the head (Fig. 5), suggesting that large deletions in the cluster had an impact on maintenance. Accordingly, Del9 fetuses showed strong reporter activity throughout the embryos, in contrast to the pattern observed three days earlier (Fig. 5). This progressive de- regulation of maintenance along with size-reduction of the cluster indicated that qualitative as well as quantitative parameters are involved in this process. At the same stage, expression of a knock-in Hoxd1/lac allele was absent from most structures that showed nonmaintained Hoxd11/lac expression in Del9 (Fig. 5;right), suggesting that the inability of Del9 to maintain expression correctly correlated with the absence of the cluster rather than with the position of the reporter gene.

Figure 4. Hoxd11/lacZ gene expression in HoxD deletions. (Left to right) TgH[d11/lac]Ge, Del3, Del7, and Del9 embryos. (A–D) In E9 embryos, the Discussion TgH[d11/lac]Ge locus was expressed around the proctodeum (A), like the neighboring Hoxd13 gene. The Del3 allele showed a more anterior domain, Human HOXD genes in transgenic mice mimicking Hoxd11 (B). The brackets emphasize the absence of staining in limb buds. In the right two panels (C,D), the Del7 and Del9 alleles showed Mice with a human HOXD complex expressed expression profiles expanded anteriorly, involving the forelimb buds. (E–H) these genes during trunk development with the In E11 embryos, the TgH[d11/lac]Ge locus (E) showed the most posterior expected specificity, showing that control expression. In the trunk, the anterior limit was located at pre-vertebra 27, mechanisms responsible for determining both the future lumbo–sacral transition (black arrowhead in E–H). Blue arrow- the tissue type and positioning of the expression heads indicate expression in digit primordia. In Del3 embryos, the limit of boundaries are identical in mouse and human expression was at pre-vertebra 25 (F). Expression in hernial gut (F, red ar- (Tuggle et al. 1990). Expression domains of hu- rowhead) was maintained in the other configurations to the right (G–H). In man genes, such as HOXD11, were established Del7 embryos, the anterior limit of expression in the central nervous sys- with appropriate anterior boundaries, even when tem was reminiscent of Hoxd3 (G). In Del9 embryos, few cells only showed corresponding mouse endogenous functions were expression in either paraxial mesoderm or spinal cord (H). The strong stain- abrogated, and human products could restore a ing in cervical pre-vertebrae detected from the Del7 locus was lost. In ven- normal vertebral column, indicating that auto- tral regions such as the hyoid, rib primordia, and ventral tail mesoderm, and cross-regulatory interactions (Maconochie et expression was seen as anterior as the first branchial arch. Expression in al. 1997) between HOXD proteins were not likely both the digit primordia and hernial gut were preserved in the absence of the behind this observation. However, the exact role HoxD cluster (H). of paralogous genes from other Hox clusters in this process will have to be determined. Rescue was not unexpected as protein se- the three major sites of transgene expression in the Del3 quences are essentially identical between mouse and hu- and Del7 loci, besides the developing trunk (i.e., the dis- man. However, mice rescued by the human complex of- tal limbs, genital eminence, and intestinal hernia), still ten showed five lumbar vertebrae instead of six, even stained strongly, even though none of these expression though expression of the human genes was like that of specificities was detected on random integration of the the mouse counterparts. A given Hox expression bound- same transgene. From these deletions, we confirmed that ary is often defined as the body level wherein transcrip- while the presence of the complex is required for the tion becomes robust. Nonetheless, low levels of tran- colinear process in the trunk, regulatory elements re- scripts are routinely found immediately anterior to such sponsible for additional expression features were located defined boundaries. Therefore, overexpression of a gene mostly outside the cluster. in its normal expression domain may increase the level of transcript to reach a functional threshold in an ante- Maintenance of expression rior adjacent metamere. In this view, a quantitative difference would be translated into an anterior shift of the Once Hox genes have been activated, their expression functional domain (i.e., a mere increased quantity of nor- needs to be maintained (Deschamps et al. 1999). We used mally expressed transcripts may lead to an apparent our set of deletions to assess the importance of the clus- homeotic transformation;Charité et al. 1995). ter in this mechanism by looking for expression mainte- In contrast to the trunk, the human HOXD complex nance of the same transgene within a Hox complex pro- was unable to rescue the limb phenotype induced by the gressively reduced in size. At 12.5 days, the patterns deletion of Hoxd11, Hoxd12, and Hoxd13, due to the were maintained and staining was absent from the heads absence of human transcripts in the developing pre- of all four fetuses (Fig. 4E-H). At 15.5 days, both the sumptive digit area. Previous work suggested that an en- TgH[d11/lac]Ge and Del3 embryos showed a tight restric- hancer sequence responsible for expression of mouse tion of the expression patterns to the posterior parts, Hoxd genes in digits was located outside the cluster, indicating maintenance of the early pattern (Fig. 5). upstream of Hoxd13. Our human PAC contained 40 kb

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function of this gene family, elaborated through the amplification of a few original genes, which were then used to specify anterior to posterior information in a colinear fashion. This ancestral regulatory module was maintained in most meta- zoans displaying a rostral–caudal axis and bilateral symmetry. Subsequently, subsets of these genes were recruited for novel functions, through changes either in cis-DNA or in those factors Figure 5. Maintenance of Hoxd gene expression. (Left to right) TgH[d11/ that could recognize a preexisting cis-acting lac]Ge, Del3, Del7, and Del9 E15 fetuses are shown. Both TgH[d11/lac]Ge DNA sequence turning it into an active control and Del3 fetuses showed tight maintenance of the Hoxd11/lacZ expression element. The fact that Hox clusters are tightly pattern at E11. Cells anterior to the early expression boundaries showed no organized, with a gene about every 10 kb and em- staining at a later stage. In Del7 late fetuses, however, some diffuse staining bedded regulatory elements, suggests that the appeared in anterior regions, i.e., the early pattern was no longer main- emergence of these enhancers from inside the tained. In Del9 fetuses, expression spread throughout the entire specimen, cluster might have disturbed the ongoing ancient indicating a deficient maintenance. The right panel (d1/lacZ) shows that gene regulatory circuitry with pleiotropic conse- expression at the Hoxd1 locus in a fully preserved complex was maintained quences. as well, indicating that the nonmaintenance of the Del9 configuration was not caused by sequences in 3Ј of the Hoxd1 locus, but rather to the deletion Maintenance of expression the cluster. In Drosophila, expression of Hox genes follows of DNA upstream of HOXD13, yet posterior genes were two phases. After their activation, transcription is main- silent in digits, indicating that this enhancer was located tained via the action of activating and repressing factors. further upstream from the cluster. The minimal distance The Polycomb group of genes (Pc-G) maintains genes was extended to 100 kb after the mouse BAC transgene silenced wherever they have not been activated, there- experiment. Interestingly, human posterior genes were fore, Pc-G loss-of-function mutations derepress Hox expressed transiently in an early phase of limb bud de- gene transcription. Pc-G-mediated repression is achieved velopment, with a posterior restriction. This particular through the interaction between a protein complex and phase was observed during the budding of zebrafish pec- Polycomb response elements (Pirrotta 1999). Inactiva- toral fins and was hypothesized to be an ancient feature tion of murine Pc-like genes suggested that vertebrate of Hox gene expression in appendices, perhaps related to Hox gene expression may also rely on such a system (e.g., their expression in the trunk (Sordino et al. 1995). The Gould 1997). fact that an isolated human HOXD complex could repro- Here, we show that expression of a Hox reporter trans- duce this trait further suggests that the early and tran- gene is maintained differentially depending on the pres- sient posterior expression in appendages is linked to the ence or absence of the surrounding complex. Tight main- cluster itself, rather than to sequences that developed tenance was observed when most of the cluster was pres- subsequently during tetrapod evolution. ent, whereas maintenance was abolished when the cluster was reduced in size. Interestingly, deletion of two- Deletion of the HoxD complex thirds of the cluster gave an intermediate picture with a somewhat reduced maintenance ability. This progres- A targeted HoxD deletion and its replacement with a sive loss of maintenance may indicate an additive effect Hoxd11/lacZ reporter gene confirmed and extended the of multiple sites in operating the silencing process. results obtained through the transgenic approach. Mice carrying this deficiency showed phenotypic alterations resulting mostly from the combination of single loss-of- Materials and methods function alleles;therefore, the phenotype was somewhat Transgenic mice, ES cells, and targeted deletions related to the Del7 mutant condition (Zakany and The human PAC78J1 was obtained using a PAC library (Genome Sys- Duboule 1999). Upon deletion of the cluster, modifica- tems;catalog no. FPAC-3387) and a human HOXD9 probe. Restriction tions in the expression pattern of the reporter construct analysis and hybridization showed that this PAC contained almost the were seen. However, expression in limbs was main- entire HOXD complex, from 30 kb upstream of EVX2 to 10 kb down- tained. Prior observations on random integration of stream of HOXD3. The PAC was linearized with Sgf1 and injected in C57BL/6 × DBA F fertilized mouse eggs as in Schedl et al. (1993). G transgenes away from the HoxD locus, as well as the 1 0 mice were screened by Southern blot with human probes specific for inability of the PAC and BAC transgenes to implement HOXD13 and the HOXD8 to HOXD4 intergenic region. The integrity of this regulation, position these regulations outside the the PAC was verified using several probes and by PCR. Copy number was cluster. estimated by comparison with a mouse HoxD probe. The expression of the Hoxd11/lacZ reporter transgene, ES cell culture, electroporation, chimera production, skeletal prepara- after deletion of the cluster, was complementary to that tions and X-gal staining were as described previously (van der Hoeven et observed with the human PAC transgene, such that al. 1996). ES cells carrying the TgH[d11/lac]Ge allele (van der Hoeven et summation of both profiles would produce a pan-Hox al. 1996) were targeted in further experiments to introduce a second loxP pattern, similar to the Del7 configuration (Zakany and site (Del3, Zakany and Duboule 1996;Del7, Zakany and Duboule 1999; loxP Duboule 1999). Consequently, Hox expression domains Del9, this study). To produce this latter chromosome, a site was introduced into a HindIII site in the second exon of Hoxd1 (Frohman and can be separated between those controlled by sequences Martin 1992) with the same polarity as the loxP site in the TgH[d11/ located within the complex itself and those depending lac]Ge ES cell. Clones from secondary targeting event were treated with on enhancer sequences located distal to the cluster. In Cre recombinase to induce deletions, which were passed through the the first case, these controls likely illustrate the ancient germ line of mice.

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BAC recombination and transgenesis Gu, H., Zou, Y. R., and Rajewsky, K. 1993. Independent control of im- A mouse BAC (RPCI23–400h17) was identified in the GenBank database munoglobulin switch recombination at individual switch regions and obtained from Roswell Park Cancer Institute (Buffalo, NY). To in- evidenced through Cre–loxP-mediated gene targeting. Cell 73: 1155– troduce LacZ reporter sequences into the Hoxd11 gene, we used ET 1164. recombination (Zhang et al. 1998;Muyrers et al. 1999;Nefedov et al. Hérault, Y., Beckers, J., Kondo, T., Fraudeau, N., and Duboule, D. 1998. 2000). The kanamycin resistance gene from pUC4Km (gift of S. Lin, Genetic analysis of a Hoxd-12 regulatory element reveals global ver- Medical College of Georgia, Altanta) was inserted downstream of the sus local modes of controls in the HoxD complex. Development 125: polyadenation sequence of the LacZ reporter gene, in an Afl2–Avr2 sub- 1669–1677. clone of the pGemE/ElacZpA construct (Gérard et al. 1993). The result- Hérault, Y., Beckers, J., Gérard, M., and Duboule, D. 1999. Hox gene ing plasmid was digested by NcoI and PpuMI and the 5.8 kb Hoxd11/ expression in limbs: Colinearity by opposite regulatory controls. Dev. lacZpAKanR fragment purified by gel electrophoresis. This targeting Biol. 208: 157–165. fragment was electroporated in DH10B cells containing both the target Kmita, M., Kondo, T., and Duboule, D. 2000a. Targeted inversion of a BAC and the pGETrec plasmid, after 40 min induction with 0.2% (w/v) polar silencer within the HoxD complex re-allocates domains of en- L-arabinose. Eleven recombinant colonies that grew on 12.5 µg/mL chlor- hancer sharing. Nat. Genet. 26: 451–454. amphenicol and 20 µg/mL kanamycin were analyzed. All had the correct Kmita, M., van der Hoeven, F., Zakany, J., Krumlauf, R., and Duboule, D. integration of lacZ in Hoxd11. The integrity of the BACs was verified by 2000b. Mechanisms of Hox gene colinearity: Transposition of the restriction enzyme fingerprinting with EcoRI, HincII, HindIII, and XhoI. anterior Hoxb1 gene into the posterior HoxD complex. Genes & Dev. No change between the original 400h17 BAC and the 400h17–d11/lac 14: 198–211. BAC was detected, except those caused by the lacZ insertion. The BAC Kondo, T. and Duboule, D. 1999. Breaking colinearity in the mouse was linearized by PI–SceI and injected as for the PAC. HoxD complex. Cell 97: 407–417. Krumlauf, R. 1994. Hox genes in vertebrate development. Cell 78: 191– Whole-mount in situ hybridization 201. WISH was performed using human probes derived from the 3Ј UTRs of Maconochie, M.K., Nonchev, S., Studer, M., Chan, S.K., Popperl, H., HOXD13, HOXD11, and HOXD9. The HOXD13 antisense probe was Sham, M.H., Mann, R., and Krumlauf, R. 1997. Cross-regulation in obtained from the EST nj13h05 and the HOXD11 probe from EST the mouse HoxB complex: The expression of Hoxb2 in rhombomere nh27c09. 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Large scale transgenic and cluster deletion analysis of the HoxD complex separate an ancestral regulatory module from evolutionary innovations

François Spitz, Federico Gonzalez, Catherine Peichel, et al.

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